In this reporting period we published or obtained preliminary data about the following related to biomarkers of catecholaminergic neurodegeneration. (1) Alpha-synuclein (AS) deposition in sympathetic ganglion tissue characterizes Lewy body forms of neurogenic orthostatic hypotension (nOH): Using immunofluorescence confocal microscopy for AS and tyrosine hydroxylase (TH, an index of sympathetic noradrenergic innervation) we described qualitatively a spectrum of abnormalities of ganglionic AS and TH in chronic autonomic failure syndromes (Isonaka et al., Clin Auton Res 2018;28:223-230). We also noted that Lewy bodies contain a core of TH surrounded by a rim of colocalized AS with TH. (2) AS deposition in sympathetic noradrenergic nerves in skin biopsies is associated with neuroimaging evidence of cardiac noradrenergic deficiency in Lewy body forms of nOH: We conducted immunofluorescence microscopy for AS, TH, and AS-TH colocalization in skin biopsies from patients with nOH. We found that increased AS-TH colocalization in sympathetic noradrenergically innervated skin constituents efficiently separates Lewy body from non-Lewy body forms of nOH and is associated with cardiac sympathetic neuroimaging evidence of myocardial norepinephrine deficiency (Isonaka et al., Hypertension 2019;73:910-918). (3) Biomarkers of central dopamine and cardiac norepinephrine deficiency predict PD in at-risk individuals: In people with multiple PD risk factors (family history, olfactory dysfunction, dream enactment, OH), those with low cerebrospinal fluid levels of endogenous DOPA (the immediate product of the rate-limiting enzyme in dopamine biosynthesis) and low levels of 3,4-dihydroxyphenylacetic acid (the main neuronal metabolite of dopamine) develop clinical disease during follow-up (Goldstein et al., Park Rel Dis 2018;50:108-112). Also in at-risk individuals, neuroimaging evidence of cardiac noradrenergic deficiency predicts PD (Goldstein et al., Park Rel Dis 2018;52:90-93). (4) AS is colocalized with TH in submandibular gland tissue in PD: We have assayed post-mortem submandibular gland tissue from patients with PD and controls. We have found preliminarily that PD entails increased AS-TH colocalization in submandibular gland tissue. Unexpectedly, catecholamine contents in the same tissues seem normal, suggesting that in submandibular gland tissue AS deposition in sympathetic noradrenergic nerves is a non-pathogenic biomarker (Isonaka et al., unpublished observations). (5) Sick-before-dead phenomenon in synucleinopathies. Lewy body forms of alpha-synucleinopathy involve decreased cardiac sympathetic innervation and functional abnormalities in residual myocardial noradrenergic terminals. We carried out an observational, retrospective, cohort study about long-term trends in indices of cardiac sympathetic innervation and function in synucleinopathies. We found that in PD patients without OH, cardiac sympathetic innervation as measured by myocardial uptake of 18F-dopamine declined at a median rate of 4.1% per year. There was no trend over years in the rate of loss of radioactivity after 18F-dopamine uptake; instead, even upon initial testing the rate of loss of radioactivity after 18F-dopamine uptake was already increased. The results are consistent with a functional abnormalitydecreased vesicular sequestration of cytoplasmic catecholaminespreceding loss cardiac sympathetic noradrenergic neurons. We call this the sick-before-dead phenomenon (Lamotte et al., unpublished observations). (6) Collaborations: (a) PD genotype-phenotype relationships: In collaboration with Dr. Derek Narendra we are exploring genotype-phenotype relationships in individuals at genetic risk of PD. In this reporting period we published evidence that in PD from DJ-1 mutation (PARK7) there is AS deposition within sympathetic noradrenergic nerves (Narendra et al., Neurology 2019;92:1113-1115). Also, we have found preliminarily that there is increased AS-TH colocalization in sympathetic nerves in PARK1 (from A53T mutation of the gene encoding AS), PARK4 (AS gene duplication), and glucocerebrosidase gene mutation but not in PARK2 (from parkin gene mutation (Isonaka et al., unpublished observations). The latter is not considered to be a Lewy body disease in most patients. (b) Relationship of AS deposition in pilomotor muscles with cardiac noradrenergic deficiency in autonomic synucleinopathies: In a collaborative study with investigators at Harvard we have found preliminarily that there is an association between innervation-adjusted AS in arrector pili muscles and cardiac noradrenergic deficiency in autonomic synucleinopathies (Isonaka et al., unpublished observations). (c) Attenuated heart rate increase during hypotension is a biomarker of nOH. In collaboration with investigators at NYU as part of the Autonomic Rare Diseases Clinical Research Consortium we published that a blunted heart rate increase suggests a neurogenic cause of orthostatic hypotension (Norcliffe-Kaufmann et al., Ann Neurol 2018;83:522-531).